Chromosome topology underlying factors: studies on a model gene locus and an exemplary DNA looping protein

Abstract

Nuclear organization is an important factor that can be a contributing factor to the function of the genome, transcription. Nuclear organization is a relatively topic in that is a mechanism to regulate transcription of genes. It describes chromosomal organization at the level of the position of genomic loci relative to nuclear structures that are associated with repressive or permissive transcriptional states. On a smaller scale, nuclear organization describes chromatin organization of topological domains or at the level of enhancer-promoter interactions. In the first chapter, the role of the position of genes in the nucleus relative to their function is reviewed. It is apparent that repressive and permissive regions in the nucleus exist that are mostly associated with silencing or transcription of genes, respectively. In the second chapter, a well-known protein is reviewed that is involved in functions of the genome by binding to it and setting up interactions between genomic loci. CTCF binding is conserved through many species and cell types and binds mostly to gene rich regions, inside gene bodies but also at intergenic regions. Cohesin, an important binding partner of CTCF shares many binding sites with CTCF in the genome suggesting this co-association has a function. Nevertheless, both proteins bind the genome independently of each other. The complex binding pattern of CTCF shows that it can be involved in many processes in the cell, including the formation of chromatin loops. What the function of nuclear organization has for transcription? This is a big question in the field of transcriptional regulation. In the third chapter I try to answer this question using a famous gene model, the immunoglobulin heavy chain (IgH) locus. IgH is part of the B cell receptor and is important for the adaptive immune response. This protein is synthesized from the productive allele, whereas the other allele, the non-productive allele, does not produce protein. We describe an allele specific 4C sequencing method that allowed the separate analysis of the alleles by using the single nucleotide polymorphisms (SNPs) between the two alleles. We found that both alleles, despite the difference in productivity do not adopt a different nuclear organization. Furthermore, we provide proof for the fact that peri-centromeric recruitment of the non-productive allele is not necessary for its allelic exclusion in mature B cells. In the fifth chapter we extensively study the role of CTCF in mediating long-range chromatin loops in a tissue specific manner. We have characterized many CTCF binding sites (CTCFbs) and have assessed their involvement in chromatin loops. We classify CTCFbs associated with cohesion and / or the enhancer mark H3K27ac. These classes of CTCFbs preferentially show homo-typic interactions, meaning that they mainly interact with CTCFbs of their own class. Furthermore, we find that conserved CTCFbs surrounding tissue specific genes are often involved in tissue specific interactions through association with tissue specific enhancer marks. This opens the interesting possibility that CTCF associates with tissue specific factors to induce tissue specific chromatin loops that possibly influence the transcription of these genes.